Anti-virulence Agents Research Articles

MarA, SoxS and Rob of Escherichia coli - Global regulators of multidrug resistance, virulence and stress response.

Bacteria have a great capacity for adjusting their metabolism in response to environmental changes by linking extracellular stimuli to the regulation of genes by transcription factors. By working in a co-operative manner, transcription factors provide a rapid response to external threats, allowing the bacteria to survive. This review will focus on transcription factors MarA, SoxS and Rob in Escherichia coli, three members of the AraC family of proteins. These homologous proteins exemplify the ability to respond to multiple threats such as oxidative stress, drugs and toxic compounds, acidic pH, and host antimicrobial peptides. MarA, SoxS and Rob recognize similar DNA sequences in the promoter region of more than 40 regulatory target genes. As their regulons overlap, a finely tuned adaptive response allows E. coli to survive in the presence of different assaults in a co-ordinated manner. These regulators are well conserved amongst Enterobacteriaceae and due to their broad involvement in bacterial adaptation in the host, have recently been explored as targets to develop new anti-virulence agents. The regulators are also being examined for their roles in novel technologies such as biofuel production.

Design, synthesis, inhibition studies, and molecular modeling of pepstatin analogues addressing different secreted aspartic proteinases of Candida albicans

The family of secreted aspartic proteinases is known as an important virulence factor of yeast infections by Candida albicans in particular, which is the most common fungal pathogen for humans with respect to systemic disease. Due to the continuing increase of drug resistant strains, these proteinases are currently considered as promising drug target candidates. Based on the known Sap2-substrate specificity data and X-ray analyses of Sap/inhibitor complexes, three libraries of inhibitors were designed and synthesized by modifying the structure of pepstatin A, a common non-selective aspartic proteinase inhibitor, at the P3, P2, or P2′ position. These novel inhibitors showed high inhibitory potencies for the isoenzymes Sap1, Sap3, Sap5 and Sap6. Then, the affinity and selectivity of the peptide ligands were investigated by molecular modeling, highlighting new key structural information for the design of potent and selective anti-virulence agents targeting Candida albicans.

Role of the CAI-1 Fatty Acid Tail in the Vibrio cholerae Quorum Sensing Response

Quorum sensing is a mechanism of chemical communication among bacteria that enables collective behaviors. In V. cholerae, the etiological agent of the disease cholera, quorum sensing controls group behaviors including virulence factor production and biofilm formation. The major V. cholerae quorum-sensing system consists of the extracellular signal molecule called CAI-1 and its cognate membrane bound receptor called CqsS. Here, the ligand binding activity of CqsS is probed with structural analogues of the natural signal. Enabled by our discovery of a structurally simplified analogue of CAI-1, we prepared and analyzed a focused library. The molecules were designed to probe the effects of conformational and structural changes along the length of the fatty acid tail of CAI-1. Our results, combined with pharmacophore modeling, suggest a molecular basis for signal molecule recognition and receptor fidelity with respect to the fatty acid tail portion of CAI-1. These efforts provide novel probes to enhance discovery of antivirulence agents for the treatment of V. cholerae.

Flavone Reduces the Production of Virulence Factors, Staphyloxanthin and α-Hemolysin, in Staphylococcus aureus

Staphylococcus aureus is a leading cause of nosocomial infections due to its resistance to diverse antibiotics. This bacterium produces a large number of extracellular virulence factors that are closely associated with specific diseases. In this study, diverse plant flavonoids were investigated to identify a novel anti-virulence compound against two S. aureus strains. Flavone, a backbone compound of flavonoids, at subinhibitory concentration (50 μg/mL), markedly reduced the production of staphyloxanthin and α-hemolysin. This staphyloxanthin reduction rendered the S. aureus cells 100 times more vulnerable to hydrogen peroxide in the presence of flavone. In addition, flavone significantly decreased the hemolysis of human red blood by S. aureus, and the transcriptional level of α-hemolysin gene hla and a global regulator gene sae in S. aureus cells. This finding supported the usefulness of flavone as a potential antivirulence agent against antibiotic-resistant S. aureus.

Structure-based virtual screening for plant-derived SdiA-selective ligands as potential antivirulent agents against uropathogenic Escherichia coli

The uropathogenic Escherichia coli pathogenecity is affected by quorum sensing transcriptional regulator SdiA. In this study, in vitro characterization of the active principles that could potentially antagonize with SdiA from the Melia dubia bark extracts has been described. After in vitro assays carried out to evaluate the inhibitory activities against the uropathogenic E. coli, the ethanolic extract (30 mg/ml) which showed the strongest suppression of haemolysis, swarming motility, hydrophobicity and biofilm formation, was subjected to GC–MS analysis and an array of 40 unrelated compounds was identified. Docking studies was conducted to screen for plant-based SdiA inhibitors. Five hits were assessed for their binding profiles and 7-(1-bromoethyl)-3, 3-dimethyl-bicyclo [4.1.0]heptan-2-one showed 66.95% binding ability with respect to C 8HSL.

Screening for novel quorum-sensing inhibitors to interfere with the formation of Pseudomonas aeruginosa biofilm

The objective of this study was to screen for novel quorum-sensing inhibitors (QSIs) from traditional Chinese medicines (TCMs) that inhibit bacterial biofilm formation. Six of 46 active components found in TCMs were identified as putative QSIs based on molecular docking studies. Of these, three compounds inhibited biofilm formation by Pseudomonas aeruginosa and Stenotrophomonas maltophilia at a concentration of 200 µM. A fourth compound (emodin) significantly inhibited biofilm formation at 20 µM and induced proteolysis of the quorum-sensing signal receptor TraR in Escherichia coli at a concentration of 3-30 mM. Emodin also increased the activity of ampicillin against P. aeruginosa. Therefore, emodin might be suitable for development into an antivirulence and antibacterial agent.

Tomatidine acts in synergy with aminoglycoside antibiotics against multiresistant Staphylococcus aureus and prevents virulence gene expression

This study characterized the multiple biological activities of the natural compound tomatidine against Staphylococcus aureus. Notably, this work examined the antibacterial activity of tomatidine in combination with other antibiotics and the influence of this compound on the expression of virulence factors in S. aureus. The effect of tomatidine on the susceptibility of S. aureus to several antibiotic classes was determined by a broth microdilution procedure and a chequerboard protocol to measure fractional inhibitory concentration indices and to reveal drug interactions. Time-kill experiments for aminoglycoside/tomatidine combinations were also performed. The haemolytic ability of several strains in the presence of tomatidine was measured on blood agar plates and the expression of virulence-associated genes in strain ATCC 29213 treated with tomatidine was monitored by quantitative PCR. Tomatidine specifically potentiated the inhibitory effect of aminoglycosides but not of other classes of drugs. This potentiating effect was observed against strains of different clinical origins (human blood, cystic fibrosis airways, osteomyelitis, skin tissues and bovine mastitis), including aminoglycoside-resistant bacteria possessing the aac(6')-aph(2″), ant(4')-Ia and aph(3')-IIIa genes. The killing kinetics for the combination of aminoglycosides with tomatidine revealed strong bactericidal activity. Although tomatidine did not possess growth-inhibitory activity of its own against prototypical S. aureus, it inhibited the haemolytic activity of several strains and, more specifically, blocked the expression of several genes normally influenced by the agr system. These results show that tomatidine is an aminoglycoside potentiator that also acts as an anti-virulence agent targeting both antibiotic-susceptible and antibiotic-resistant S. aureus.

Antivirulence Agents Against MRSA

.Antibiotics are currently the treatment of choice for MRSA infections, but increasing resistance to these agents, coupled with the decline of commercial development of new antibiotics, has created an urgent need and a window of opportunity to introduce new treatment options. The WHO has identified antimicrobial resistance as one of the three greatest threats to human health. A call by the Infectious Disease Society of America to develop ten new antibacterial drugs by 2020 has been endorsed by the US Department of Health and Human Services and numerous other organizations in the USA and Europe

Novel agents to inhibit microbial virulence and pathogenicity

Importance of the field: The rising levels of antibiotic resistance in pathogenic microorganisms create an urgent need for new antimicrobial agents that are not affected by resistance mechanisms already present in the bacterial population. Targeting virulence is one of the alternative approaches to find new molecules to treat infections due to resistant bacteria. Novel strategies to identify these new antimicrobial agents have been reported.Areas covered in this review: A variety of virulence factors are now the target for inhibition mainly in the antibacterial field. This review focuses exclusively on the new virulence inhibitors published in patents (worldwide collection of patents filed) or in the literature since 2006.What the reader will gain: New efficacy in vitro assays have been developed allowing screening of large numbers of molecules. These inhibitors are mainly antibacterial molecules, a few natural products, peptides and antibodies. A growing number of these published studies provide results showing a proof of concept with antivirulence compounds that were able to prevent or treat an infection in vivo. Moreover, some new antivirulence agents could inhibit virulence mechanisms that are common to different related pathogenic species, extending the potential spectrum of antivirulence compounds.Take home message: The progress reported recently for antivirulence molecules at the preclinical stages should allow new classes of molecules to enter into development as new antimicrobial agents with new mechanisms of action.

High-Throughput Identification of Chemical Inhibitors of E. coli Group 2 Capsule Biogenesis as Anti-Virulence Agents

Rising antibiotic resistance among Escherichia coli, the leading cause of urinary tract infections (UTIs), has placed a new focus on molecular pathogenesis studies, aiming to identify new therapeutic targets. Anti-virulence agents are attractive as chemotherapeutics to attenuate an organism during disease but not necessarily during benign commensalism, thus decreasing the stress on beneficial microbial communities and lessening the emergence of resistance. We and others have demonstrated that the K antigen capsule of E. coli is a preeminent virulence determinant during UTI and more invasive diseases. Components of assembly and export are highly conserved among the major K antigen capsular types associated with UTI-causing E. coli and are distinct from the capsule biogenesis machinery of many commensal E. coli, making these attractive therapeutic targets. We conducted a screen for anti-capsular small molecules and identified an agent designated “C7” that blocks the production of K1 and K5 capsules, unrelated polysaccharide types among the Group 2–3 capsules. Herein lies proof-of-concept that this screen may be implemented with larger chemical libraries to identify second-generation small-molecule inhibitors of capsule biogenesis. These inhibitors will lead to a better understanding of capsule biogenesis and may represent a new class of therapeutics.

N-Hydroxybenzimidazole inhibitors of ExsA MAR transcription factor in Pseudomonas aeruginosa: In vitro anti-virulence activity and metabolic stability

ExsA is a multiple adaptational response (MAR) transcription factor, regulating the expression of a virulence determinant, the type III secretion system (T3SS) in Pseudomonas aeruginosa. Non-cytotoxic, non-antibacterial N-hydroxybenzimidazoles were identified as effective inhibitors of ExsA-DNA binding, and their potential utility as anti-virulence agents for P. aeruginosa was demonstrated in a whole cell assay. Select N-hydroxybenzimidazole inhibitors were stable in an in vitro human liver microsomal assay.

Anti-Infective Strategies of the Future: Is there Room for Species-Specific Antibacterial Agents?

Broad-spectrum antibiotics, directed against conserved bacterial targets, are the mainstay of antibacterial therapy. Increasing resistance, however, demands new strategies. Over time a number of therapeutic concepts have evolved, starting out with the use of polyclonal antisera, which were rapidly replaced by the easier to use antibiotics. Other concepts, such as immunotherapy, radioimmunotherapy, anti-virulence agents, phage therapy and others are under evaluation and often limited in application. In the discovery process of new antibiotics in the pharmaceutical industry quite a number of new agents have emerged, which exhibit a surprisingly high degree of species-specificity. None of them has been considered for development so far. Some examples from the literature which show selectivity for Helicobacter pylori, Pseudomonas aeruginosa, Haemophilus influenzae, Staphylococcus aureus, anaerobes, and others, will be discussed here. It is postulated that there is a room for such agents in future antibacterial therapy, e.g. in difficult to treat infections caused by nonfermenters such as multiresistant P. aerugoinosa, Acinetobacter, Enterobacteriaceae, and S.aureus, including MRSA. Their application would include monotherapy as well as combination therapy with other antibiotics, anti-virulence agents or immunotherapy and these possibilities would greatly expand the current anti-infective armamentarium.

Prospects for the Development and Use of Gram-Negative Bacterial Virulence Protein Secretion Inhibitors for Prophylaxis and Treatment of Infectious Diseases

As the breadth and number of bacterial pathogens exhibiting antibiotic resistance is rapidly increasing, our ability to treat new and re-emerging infectious diseases is being threatened. Therefore, the development of new therapeutic strategies, including drugs acting on new targets, is required. In this review, the feasibility of gram-negative bacterial secretion systems for the development of anti-virulence agents and possible arenas for their utility in therapeutic intervention of gram-negative bacterial infections such as gastroenteritis, nosocomial infections, and venereal diseases are discussed.

N-hydroxybenzimidazole inhibitors of the transcription factor LcrF in Yersinia: novel antivirulence agents.

LcrF, a multiple adaptational response (MAR) transcription factor, regulates virulence in Yersinia pestis and Yersinia pseudotuberculosis. In a search for small molecule inhibitors of LcrF, an acrylic amide series of N-hydroxybenzimidazoles was synthesized and the SAR (structure-activity relationship) was examined. Selected test compounds demonstrated inhibitory activity in a primary cell-free LcrF-DNA binding assay as well as in a secondary whole cell assay (type III secretion system dependent Y. pseudotuberculosis cytotoxicity assay). The inhibitors exhibited no measurable antibacterial activity in vitro, confirming that they do not target bacterial growth. These results demonstrate that N-hydroxybenzimidazole inhibitors, exemplified by 14, 22, and 36, are effective antivirulence agents and have the potential to prevent infections caused by Yersinia spp.

Directed Evolution of a Quorum-Quenching Lactonase from Mycobacterium avium subsp. paratuberculosis K-10 in the Amidohydrolase Superfamily

The PLL(PTE-like lactonase)-group of enzymes within the amidohydrolase superfamily hydrolyze N-acyl-homoserine lactones (AHLs) that are involved in bacterial quorum-sensing pathways. These enzymes possess the (beta/alpha)(8)-barrel fold and serve as attractive templates for in vitro evolution and engineering of quorum-quenching biological molecules that can serve as antivirulence therapeutic agents. Using a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 (GI: 41409766) as the initial template for in vitro evolution experiments, we enhanced the catalytic efficiency and increased the substrate range of the wild-type enzyme through a single point mutation on the loop at the C-terminal end of the eighth beta-strand. This N266Y mutant had an increased value of k(cat)/K(M) of 30- and 32-fold toward 3-oxo-N-octanoyl-l-homoserine lactone and N-hexanoyl-l-homoserine lactone, respectively; the evolved mutant also exhibited lactonase activity toward 3-oxo-N-hexanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone, AHLs that were previously not hydrolyzed by the wild-type enzyme. This article reinforces the evolutionary potential of the (beta/alpha)(8)-barrel fold and highlights the possibility of using quorum-quenching lactonases in the amidohydrolase superfamily as templates for engineering biomolecules of therapeutic use.

Probing of the cis-5-phenyl proline scaffold as a platform for the synthesis of mechanism-based inhibitors of the Staphylococcus aureus sortase SrtA isoform

cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile. 4-Vinylsulfonyl 5-phenyl prolinates inhibit Staphylococcus aureus sortase SrtA irreversibly by modification of the enzyme Cys184 and could be used as hits for the development of antibacterials and antivirulence agents.

Adjunct antimicrobial therapy – prospects for the future

The bacterium is a relatively simple microorganism compared with mammalian cells, generally ranging in size from just under 1200 to over 6000 genes, but has evolved an amazing repertoire of defenses against environmental insult, toxins and other microbial life. The standard of care has been to kill the bacterium when found in the host as a pathogen, and a large arsenal of drugs has been developed to do just that. However, the adaptability of these pathogens to evade the host immune system defenses and classical antibiotics has presented an evolving scenario of continuous emergence of resistance against virtually all antibiotic drug classes. It may be time to explore other options in this infection battle through the use of adjunct therapy such as immunotherapeutics and antivirulence agents.

Corneal ulceration in pediatric patients: a brief overview of progress in topical treatment.

Pediatric microbial keratitis is a rare but potentially devastating condition. The condition is similar to adult microbial keratitis, but is often characterized by a more severe inflammatory response. The micro-organisms that cause microbial keratitis in children are similar to the causative agents in adults, with herpes simplex and bacteria being the predominant causative agents, and fungi being less frequent. Of the bacterial pathogens, Pseudomonas aeruginosa, Staphylococcus aureus and alpha-hemolytic streptococci are common. The risk factors for pediatric keratitis include colonization of the eyes during birth and trauma to the cornea. Certain microbial factors involved in microbial keratitis are common to all micro-organisms, including adhesion to the cornea, penetration into the cornea, destruction of the corneal stroma (usually by microbial and/or host proteases), and recruitment of white blood cells to help defend the eye. Specific inflammatory responses that occur during pediatric microbial keratitis are not known in detail, but it is likely that cytokines and polymorphonuclear leucocytes are major factors, as they are in adult microbial keratitis. Treatment for pediatric microbial keratitis is usually the same as treatment for adult microbial keratitis; topical application of antimicrobial agents initially, followed by application of anti-inflammatory agents. With pediatric microbial keratitis, extra care must be taken to ensure nontoxicity due to blood adsorption. New microbial keratitis treatments are being developed and these mainly focus on new antimicrobials, antivirulence agents (such as vaccination against microbial toxins) or specific anti-inflammatory agents. There remains a clear need for increased research into the specific responses during microbial keratitis in children which will help progress new therapies as well as the development of new antimicrobials, especially new antifungal therapies.